Suppression of IL‐8‐Src signalling axis by 17β‐estradiol inhibits human mesenchymal stem cells‐mediated gastric cancer invasion

Abstract Epidemiologic data show the incidence of gastric cancer in men is twofold higher than in women worldwide. Oestrogen is reported to have the capacity against gastric cancer development. Endogenous oestrogen reduces gastric cancer incidence in women. Cancer patients treated with oestrogens have a lower subsequent risk of gastric cancer. Accumulating studies report that bone marrow mesenchymal stem cells (BMMSCs) might contribute to the progression of gastric cancer through paracrine effect of soluble factors. Here, we further explore the effect of oestrogen on BMMSCs‐mediated human gastric cancer invasive motility. We founded that HBMMSCs notably secrete interleukin‐8 (IL‐8) protein. Administration of IL‐8 specific neutralizing antibody significantly inhibits HBMMSCs‐mediated gastric cancer motility. Treatment of recombinant IL‐8 soluble protein confirmed the role of IL‐8 in mediating HBMMSCs‐up‐regulated cell motility. IL‐8 up‐regulates motility activity through Src signalling pathway in human gastric cancer. We further observed that 17β ‐estradiol inhibit HBMMSCS‐induced cell motility via suppressing activation of IL8‐Src signalling in human gastric cancer cells. 17β‐estradiol inhibits IL8‐up‐regulated Src downstream target proteins including p‐Cas, p‐paxillin, p‐ERK1/2, p‐JNK1/2, MMP9, tPA and uPA. These results suggest that 17β‐estradiol significantly inhibits HBMMSCS‐induced invasive motility through suppressing IL8‐Src signalling axis in human gastric cancer cells.


Introduction
Mesenchymal stem cells (MSCs) as an important population of cells that contribute to tumour progression and drug resistance [1,2].
Mesenchymal stem cells can migrate to the damaged tissue and the microenvironment of developing gastric tumour [3], in which MSCs might promote tumour malignance through tumour growth, invasion, metastasis formation and therapy resistance, through paracrine secretion of cytokines, chemokines, growth factors and the differentiation of MSCs into tumour-associated fibroblasts [1,[4][5][6]. So far, gastric carcinoma is one of the most prevalent cancers worldwide and is the secondary leading cause of cancer-related mortality [7] because of the poor prognosis of patients. Invasion and metastasis are the major causes of death from human gastric cancer.
Interleukin-8 (IL-8 or called CXCL8), a cytokine of the CXC chemokine family, has been proposed to contribute to chronic inflammation and cancer development. Human mesenchymal stem cells (hMSCs) enhance cell motility of human HLaC78 head and neck squamous cell carcinoma cell line and IL-8 cytokine secretion [8]. High expression of IL-8 in adipose-derived stem cells (ASCs) may support breast tumour growth and progression [9]. Interleukin-8 may play a critical role in regulating the progressive growth of human gastric carcinoma cells [10]. Overexpression of IL-8 promotes the capacity of cellular adhesion, migration, invasion and chemoresistance in the gastric cancer cells [11]. These findings suggest that IL-8 might be a therapeutic target for gastrointestinal cancers.
Src family kinases play key roles in the development and progression of gastric cancer due of the elevated Src kinase activity in human gastric carcinoma tissues [12]. Src-dependent pathway regulates chemotaxis in response to a IL-8 gradient in neutrophils [13,14]. Src family kinases can affect cell adhesion and migration through regulating activation of Cas and paxillin scaffold proteins [15]. Studies reported that deregulation of Cas signalling is involved in stomach cancer progression and developmental defects in human [16]. Paxillin that is demonstrated as a key regulator of the cellular migration machinery 2-and 3-dimensional microenvironments has been recently regarded as the future prognostic and therapeutic target [17]. Paxillin is activated and regulates normal neutrophil adhesion and motility when IL-8 stimulation [18]. Src/extracellularly responsive kinase (ERK) signalling is reported to be associated with gastric cancer progression [19,20]. Activation of c-Jun N-terminal kinase by N-myc downstream-regulated gene 1 (NDRG1) promotes tumour angiogenesis via VEGF-A expression in gastric cancer [21]. Thus, therapeutic strategies targeting Src hold promise for the treatment of gastric cancer.
Oestrogen against gastric cancer development has been reported such as that cancer patients treated with oestrogens have a lower subsequent risk of gastric cancer, and that the delayed menopause is associated with a reduced risk for gastric cancer development [22,23]. Hormone replacement therapy (HRT) has been reported protect against gastric cancer in women, even in men [24,25]. In the animal models of N-methyl-N 0 -nitro-nitrosogunidine-induced gastric cancer, evidences also show a reduced incidence in female compared with male rats [26,27]. Oestrogen has been shown to impair CCL5-and IL6-induced gastric cancer cell motility [28,29], and reduce gastric cancer progression through suppression of erbB-2 oncogene activation [24]. These findings suggested that oestrogen might possess the protective effect against gastric cancer development. Oestrogen treatment is also reported to inhibit cell migration in human LoVo colon cancer cells [30], and attenuate hepatocellular carcinoma progression in vitro and in vivo [31].
In the present study, we investigated if 17b-estradiol (E2) treatment is sufficient to inhibit human bone marrow mesenchymal stem cells (HBMMSCs)-mediated invasive motility activity through impairing IL-8 function, and further identified the related precise molecular and cellular mechanisms in human gastric cancer cells. The results demonstrated that 17b-estradiol treatment inhibit HBMMSCSinduced cellular motility by suppressing activation and expression of IL-8-up-regulated Src downstream target proteins including phospho-Cas, phospho-paxillin, phospho-ERK1/2, phospho-JNK1/2, MMP9, tPA and uPA in human gastric cancer cells. This study suggests that 17b-estradiol possesses the properties of anti-cancer by inhibiting HBMMSCS-mediated motility activity in human gastric cancer cells.

Human cytokine array
Cell culture supernates form HBMMSCs were collected by centrifuging at 10,000 9 g for 10 min. to remove cell debris. The membranes from human protein cytokine array kit (Proteome Profiler TM Array; R&D Systems) were blocked with blocking buffer at room temperature for one hour, and were followed by incubation with cell culture supernates overnight at 2-8°C on a rocking platform. The membranes were then washed with wash buffer, and incubated with the diluted Streptavidin-HRP for 30 min. at room temperature. After washing, the membranes were assayed by chemiluminescence.

Immunoblotting
To isolate total proteins, cells were washed with cold PBS and resuspended in lysis buffer (50 mM Tris, pH 7.5, 0.5 M NaCl, 1.0 mM EDTA, pH 7.5, 10% glycerol, 1 mM BME, 1% NP40) plus proteinase inhibitor cocktail and phosphatase inhibitor cocktail (Roche Molecular Biochemicals, Penzberg, Upper Bavaria, Germany). After incubation for 30 min. on ice, the supernatant was collected by centrifugation at 12,000 9 g for 15 min. at 4°C, and the protein concentration was determined by the Bradford method. Sample containing equal proteins (40 lg) were loaded and analysed by immunoblotting. Briefly, proteins were separated by 12% SDS-PAGE and transferred onto PVDF membrane (Life Technologies). Membrane were blocked with blocking buffer (5% non-fat dry milk, 20 mM Tris-HCl, pH 7.6, 150 mM NaCl and 0.1% Tween 20) for at least 1 hr at room temperature. Membranes were incubated with primary antibodies in the above solution on an orbit shaker at 4°C overnight. Following primary antibody incubations, membranes were incubated with horseradish peroxidase-linked secondary antibodies (anti-rabbit, antimouse or anti-goat IgG). Antibody-bound protein bands were detected using high sensitive Immobilon Western Chemiluminescent HRP Substrate (Millipore), and photographed with Bio-Rad Chemiluminescence Imaging System (Bio-Rad Laboratories, Inc. Hercules, CA, USA).

Invasive motility assay
Cell invasive motility assay was performed with the 24-well modified Boyden chambers containing membrane filter inserts with 8-lm pores (Corning Incorporated Life Sciences, Tewksbury, MA, USA). Membrane filter inserts were pre-coated with collagen I (Sigma Chemical Co.). To detect the effect of HBMMSCs on cell motility, the lower compartment was seeded with HBMMSCs, and gastric cells were placed in the upper chamber. The assay system was filled with serum-and phenol red-free medium and incubated for 24 hrs in the presence of 17b-estradiol, recombinant IL-8 protein or IL-8 neutralizing antibody. After incubation, invasive cells on the bottom of membrane filter were fixed with 4% paraforrmaldehyde and stained with 1 lg/ml 4 0 -6-diamidine-2-phenylindole dihydrochloride (DAPI; Roche Molecular Biochemicals) for 30 min. to detect cell nucleus (blue stains) by fluorescence microscopy. The motility phenotypes were determined by counting the cells that migrated to the lower side of the filter with fluorescence microscopy at 409 and 1009 magnification respectively.

ELISA
Conditioned medium (CM) from both of HBMMSCs culture and 17bestradiol-treated HBMMSCs culture were collected by centrifuging at 10,000 9 g for 10 min. to remove cell debris. The level of IL-8 was measured using the ELISA kit obtained from R&D Systems according to the manufacturer's instructions.

Cell proliferation assay
To determine the effect of 17b-estradiol (E2) on gastric cancer cell viability, cells were treated with E2 (10 À10 , 10 À9 , 10 À8 M) for 24, 48 and 72 hrs and were subjected to 3-[4,5-dimethylthiazol-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co.) assay. The absorbance of blue formazan crystals was measured at 570 nm using an ELISA plate reader. The quantity of the formosan product was directly proportional to the number of viable cells in the culture medium. The cell viability of cells was determined according to the absorbance corrected to a background reading.

Statistical analysis
Each experiment was repeated at least three times. Results were presented as the mean AE S.D., and statistical comparisons were made using the Student's t-test. Significance was defined at the P < 0.05 or 0.01 levels.

17b-estradiol suppresses HBMMSCs-mediated cellular motility in human gastric cancer cells
The co-culture system of HBMMSCs/gastric cancer cells was used to value the influence of 17b-estradiol (E2) on HBMMSCs-induced cellular motility in gastric cancer cells. In this study, we detected the effect of 17b-estradiol (E2) on HBMMSCs-increased motility activity in human gastric cancer cells by co-culturing HBMMSCs and gastric cancer cells in the presence of E2 (10 À8 M) for 24 and 48 hrs. Subsequently, we observed the ability of motility in gastric cancer cells by motility assay. In the motility assay (Fig. 1), the findings showed that E2 (10 À8 M) notably inhibits HBMMSCs-mediated motility activity in human AGS and CS12 cells.

Analysis of secreted cytokines from HBMMSCs and human gastric cancer cells
To determine which kind of cytokines were secreted by human (HBMMSCs) and gastric cancer cells in the culture medium, we used the human protein cytokine array to measure the cell culture supernates. Human bone marrow mesenchymal stem cells alone, CS12 cells alone and CS12 cells/HBMMSCs were, respectively, cultured for 24 hrs in serum-and phenol red-free IMDM medium, samples of cell culture CM were collected for cytokine protein assay. The findings showed that HBMMSCs remarkably secreted IL-8 soluble protein ( Fig. 2A).

IL-8 neutralizing antibody inhibits HBMMSCsinduced human AGS cell motility
In this study, we found IL-8 was expressed from HBMMSCs in the highest level. To identify the effect of IL-8 secreted from HBMMSCs on cellular motility activity in human gastric cancer cells, we used the specific neutralizing antibody to eliminate the function of IL-8 cytokine. Co-culture of HBMMSCs and AGS cells were established for valuing the effect of HBMMSCs on cellular motility in human gastric cancer cells. We found that HBMMSCs significantly contributed to cellular motility activity in AGS cells. However, the HBMMSCsincreased motility activity in AGS cells was diminished when using various concentrations of IL-8 neutralizing antibody in this co-culture system (Fig. 2B). The findings suggested that IL-8 secreted from HBMMSCs plays a critical role in the induction of cell motility in human gastric cancer cells.

IL-8 promotes motility activity in human gastric cancer cells
To further confirm the role of IL-8 in increasing the motility activity in gastric cancer cells, we treated AGS and CS12 cells with various concentrations (0, 1, 10, 20, 50 and 100 ng/ml) of recombinant IL-8 for 24 hrs. Subsequently, the motility activity of gastric cancer cells was valued by cellular motility assay ( Fig. 2C and D). We observed that IL-8 in various concentrations (1, 10, 20, 50 and 100 ng/ml) induce a significant increase in cell motility in human AGS and CS12 gastric cancer cells.

17b-estradiol inhibits IL-8-induced motility in human gastric cancer cells
We explored the effect of 17b-estradiol (E2) on IL-8-up-regulated motility activity in human gastric cancer cells. We treated AGS and CS12 cells with various concentrations (0, 1, 10 and 20 ng/ml) of IL-8 soluble protein in the presence of 17b-estradiol (E2; 10 À8 M) for 24 hrs. We observed that 17b-estradiol (E2; 10 À8 M) significantly inhibits IL-8-promoted motility activity in human AGS and CS12 gastric cancer cells (Fig. 3A and B). PPT (ER-a agonist) and DPN (ER-b agonist) were also used for measure the inhibitory effect on invasive motility. The results showed the significantly inhibitory effect of PPT and DPN on invasive cell motility in human gastric cancer cells (Fig. 3C).

Discussion
Major findings of this study can be summarized as followings: (i) Results from human cytokine arrays showed that HBMMSCs remarkably secrete IL-8 soluble protein in cell culture CM. (ii) In the co-culture system of HBMMSCs/AGS cells, treatment of IL-8 specific neutralizing antibody inhibited HBMMSCs-up-regulated cell motility activity by blocking IL-8 function. The role of IL-8 in mediating cellular motility activity was further confirmed by using recombinant IL-8 soluble protein. (iii) After administration of various specific inhibitors for cellular signalling pathways, we found that Src, ERK and JKN signalling pathways mediates IL-8-up-regulated cell motility activity, and that Src mediates IL-8-induced activation of EKR and JNK signalling pathways in human gastric cancer cells. (iv) Co-culture of gastric cancer cells/HBMMSCs in the presence of 17b-estradiol (E2) showed 17b-estradiol inhibition of HBMMSCs-promoted motility activity in human gastric cancer cells. (v) Activation of Src by IL-8 might play a critical role in mediating HBMMSCs-up-regulated motility activity. However, IL-8up-regulated cell motility activity and activation of Src, Cas, paxillin, ERK1/2 and JNK1/2 were significantly inhibited by17b-estradiol (E2; 10 À8 and 10 À9 M). (vi) Furthermore, 17b-estradiol inhibits the expression of IL-8-Src signalling downstream invasive proteins MMP9, tPA and uPA in human gastric cancer cells. These results demonstrated that 17b-estradiol might inhibit HBMMSCs-mediated human gastric cancer cell motility via suppression of IL-8-Src signalling axis (Fig. 6). Mesenchymal

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CXCR4 signalling pathway [35], and MMP2 molecular factor in human medulloblastoma [36], in which subsequently may lead to the effects of angiogenesis via VEGF, MCP-1 and HIF-1 signalling pathways. High expression of IL-8 in ASCs may support breast tumour growth and progression [9]. Increased IL-8 in the bone microenvironment may represent one possible mechanism for microenvironment perversion in favour of acute lymphoblastic leukaemia cells [37]. Interleukin-8 has been proposed to contribute to chronic inflammation and cancer development. Interleukin-8 may play an important role in regulating the progressive growth of human gastric carcinoma cells [10]. Interleukin-8 overexpression increases the capacity of cellular adhesion, migration, invasion and chemoresistance in the gastric cancer cells [11]. Helicobacter pylori-increased IL-8 promotes human gastric cancer cell proliferation through trans-activation of EGF receptor (EGFR) by disintegrin and metalloproteinase (ADAM) activation [38]. In the present study, we observed HBMMSCs notably secrete IL-8 protein in the human cytokine array. Human bone marrow mesenchymal stem cells-up-regulated motility activity of AGS cells was significantly inhibited by IL-8 specific neutralizing antibody. Treatment of recombinant IL-8 confirmed the influence of IL-8 in mediating HBMMSCs-up-regulated cell motility. These findings suggested that IL-8 secreted from HBMMSCs significantly increases cellular motility activity of human gastric cancer cells via the paracrine effect. Gastric carcinogenesis is reported to be induced by active membrane-bound receptors that lead to the activation of intracellular signalling pathways through multiple GTPase proteins, receptor kinases, kinases and phosphatases. The activation of phos-  3-kinase (PI3-K)/Akt (protein kinase B) and mitogen-activated protein kinases (MAPKs) pathways occur during tumour development [39]. Mitogen-activated protein kinases include three major subfamilies such as the ERKs, the JNKs, also known as stress-activated protein kinases, and the p38 MAPKs [39]. Activation of b-catenin signalling pathway is shown to medi-ate gastric cancer growth and invasion [40]. Another pathway that has received attentions is Src pathway. Src plays a critical role in the variety of cancer cellular regulation such as cell division, motility, adhesion, angiogenesis and survival, so Src is regarded as the attractive target for future anti-cancer therapeutics. Src kinase closely regulates the phosphorylation and activation of Cas Fig. 4 17b-estradiol down-regulates IL8-induced motility activity by suppressing activation of Src, EKR and JNK signalling in human gastric cancer cells. Human gastric cancer cells were pre-treated with 17b-estradiol (10 À8 and 10 À9 M) or PP2 (Src activation inhibitor; 10 À6 and 5 9 10 À6 M) for 1 hr and followed by IL-8 (10 ng/ml) treatment for 3 hrs. AGS and CS12 cells were then harvested for immunoblotting assay. **P < 0.01 versus control (line 1); ## P < 0.01 versus IL-8 treatment (mean AE S.D., n = 3). adhesion protein and paxillin cytoskeletal protein, which operate cell migration in invasive cells during the development and pathological conditions associated cancer metastasis. Src-dependent pathway has been show to regulate neutrophils chemotaxis in response to a IL-8 gradient [13,14]. So targeting Src strategic therapies has been regarded as a great approach for gastric cancer. In this study, we observed that Src mediates IL-8-induced invasive motility in human gastric cancer cells. The critical role of Src was confirmed by using PP2, a specific inhibitor for Src, in the motility and immunoblotting assays. Compared with various signalling pathway inhibitors including LY294002 (PI3K/Akt activation inhibitor), U0126 (ERK1/2 activation inhibitor), SB203580 (p38 MAPK inhibitor), XAV939 (b-catenin inhibitor) and SP600125 (JNK1/2 inhibitor), the findings indicated that Src mainly mediates IL-8-up-regulated motility activity through modulating the activation of Cas, paxillin, EKR1/2 and JNK1/2 signalling pathways and the expression of invasive proteins MMP9, tPA and uPA in human gastric cancer cells.
Protective properties of oestrogen against gastric cancer development have been reported because of the reduced gastric cancer risk in the delayed menopausal women and in the HRT-treated population compared with non-treated one [22,23]. In the present study, we observed that 17b-estradiol treatment significantly inhibited HBMMSCS-mediated cell motility activity through suppressing IL-8-Src downstream target protein activation and expression, including Cas, paxillin, ERK1/2, JNK1/2, MMP9, tPA and uPA in human gastric cancer cells. 17b-estradiol may inhibit HBMMSCS-induced invasive motility through suppressing IL-8-Src signalling axis in human gastric cancer.

Conclusion
Gastric cancer is one of the most common cancers worldwide, and is also the second leading cause of cancer-related mortality. Accumulating evidence suggest that BMMSCs may contribute to the progression of cancer development through paracrine effect. Epidemiological data report that the incidence of gastric cancer in male is~twofold higher than in women worldwide. Studies also report the delayed menopause is associated with a reduced risk for gastric cancer development. In this study, we further observed that 17b-estradiol significantly inhibits HBMMSCS-induced invasive motility through suppressing IL-8-Src signalling axis in human gastric cancer cells. Fig. 5 The affect of 17b-estradiol on expression of IL8-Src signalling downstream invasive proteins MMP9, tPA and uPA in human gastric cancer cells. Human gastric cancer cells were pre-treated with 17b-estradiol (10 À8 M), PP2 (Src activation inhibitor; 10 À6 M), U0126 (ERK1/2 activation inhibitor; 10 À6 M) or SP600125 (JNK1/2 inhibitor; 10 À6 M) for 1 hr and followed by IL-8 (10 ng/ml) treatment for 24 hrs. AGS and CS12 cells were then harvested for immunoblotting assay.

Figure S2
The effects of 17b-estrodial on the protein expression of IL-8RΑ/Β in human gastric cancer cells.

Figure S3
The effect of E2 alone on the human gastric cancer cells motility. Figure S4 In the present study, we detected the effect of 17b-estradiol (E2) on HBMMSCs-increased motility activity in human gastric cancer cells by co-culturing HBMMSCs and gastric cancer cells in the presence of E2 (10 À8 M) for 24 hrs.